Methods and systems for metabolite and/or lipid-based detection of colorectal cancer and/or adenomatous polyps

ABSTRACT

Described herein are sets of metabolite and lipid (e.g., fatty acid) markers that can be used in the detection of early stage colorectal cancer and/or early development of adenomatous polyps. Presented herein are illustrative pathology-linked panels. In certain embodiments, the markers presented herein (or subsets thereof) are used as a panel for detecting either colorectal cancer or adenomatous polyps at the same time. The markers presented herein include metabolites and lipids (e.g., fatty acid) freely detectable and accurately quantifiable in human serum. In certain embodiments, the sample may be plasma, urine, saliva, whole blood, dried blood spot or dried serum spot.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/343,095, filed on May 30, 2016, the content of which is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates generally to methods and systems for measuringmetabolites and lipids (e.g., fatty acids) in biological samples. Inparticular embodiments, the invention relates to methods and systems fordetection of metabolites and lipids (e.g., fatty acids) in human serumor another biological sample to identify early stage colorectal cancerand/or early development of adenomatous polyps, for example, though notnecessarily, at the same time.

BACKGROUND

Digestive malignant neoplasms including esophageal, gastric, andcolorectal cancer are the most common cause of cancer induced death inthe world. For example, colorectal cancer (CRC) is the third most commoncancer in men (746,000 cases, 10.0% of total cancer cases) and thesecond most common cancer in women (614,000 cases, 9.2%) worldwide andit is the third most frequent cause of cancer mortality in the world forboth genders. The European Union alone recorded 345,000 colorectalcancer incidences and 152,000 deaths from colorectal cancer in 2012.

To decrease mortality rates, various platforms for early stage cancerdetection have been developed and tested to complete diagnosticevaluations on recruited eligible populations. However, it is difficultto cure gastrointestinal cancers detected with these platforms becausethey are often discovered at the progressive state. Moreover, themajority of the symptoms associated with gastrointestinal cancers do notmanifest themselves until late in their development. Accordingly, theperformance status of patients with gastrointestinal cancers and theiroverall prognosis needs to be improved.

For example, although screening programs for colorectal cancer havebecome more prevalent and survival rates have gone up within last 30years, only around 40-44% of the cancers are detected in an early,localized stage, likely due to the lack of sensitivity of most of thesescreening programs. The general recommendation both in the U.S. andEurope is that individuals with average risk for colorectal cancer startregular screening either with colonoscopy or fecal blood test at the ageof 50. However, the cancer incidence is increasing also among youngeradults.

Understanding alterations in metabolic profiles in the colon that occurwith tumor onset and progression could lead to better diagnostic testsas well as uncover new approaches for treatment or even prevention ofCRC. Most CRCs are believed to originate from adenomatous polyps thatacquire distinct mutations and accumulate other molecular alterationsthat allow them to progress through distinct histopathologic stagesbefore becoming invasive carcinomas. Some adenomatous polyps do notprogress to invasive tumors. Thus, the availability of a metabolicfingerprint that could distinguish a polyp that is likely to progressfrom one that will not progress could guide the frequency of screeningcolonoscopies and other preventative measures.

Metabolomics, which is understood as the quantitative measurement of all(or a certain percentage of all, e.g., most) low-molecular-weightmetabolites in an organism at a specified time under specificenvironmental conditions, has been shown to be an effective tool fordisease diagnosis, biomarker screening, and characterization ofbiological pathways.

Metabolites are the end products of cellular processes and theirconcentrations reflect the functional status of the organism and thusthey are closely related to the observed phenotype. Perturbations inbiological pathways can amplify the concentration changes ofmetabolites, making small molecule metabolites very attractivebiomarkers of disease detection.

Altered metabolism is a cancer hallmark, resulting from changes insignaling pathways, protein expression, and other molecular mechanisms.Altered metabolism also reflects specific biochemical adaptations duringcarcinogenesis, which may confer malignant cells' survival advantages.

Two of the most prominent technologies for metabolite detection andquantification are nuclear magnetic resonance (NMR) and massspectrometry (e.g., coupled to liquid chromatograph-LC-MS, gaschromatograph-GC-MS or direct analysis-DESI, DART). Both NMS and MStechnologies are widely used in research and clinical settings.

Mass spectrometry is essentially a technique for “weighing” molecules.It is based upon the motion of charged particles, called ions, in anelectric or magnetic field. The mass to charge ratio (m/z) of aparticular ion affects this motion. Since the charge of an electron isknown, then the mass to charge ratio is a measurement of an ion's mass.Mass spectrometry allows scanning for a wide range of metabolites. Datacan include several thousands of detected metabolic events and thuscreates a large pool for potential biomarker selection.

Several challenges exist in applying mass spectrometry measurements ofmetabolites and lipids in order to identify the presence or risk ofcolorectal cancer and/or adenomatous polyps in a subject.

First, the sensitivity of mass spectrometry instrumentation has onlyrecently, over the past 5 years, become sufficient to enable globalprofiling of different biomaterials. Accordingly, metabolite baseddiagnostics that look into the whole human metabolon are still in theirdevelopmental infancy.

Secondly, accurate measurements of metabolites generally requires aninternal standard for each marker of interest. Such standards formetabolites may not commercially available, and accordingly must besynthesized. Accordingly, performing accurate measurements ofmetabolites requires significantly more than just measuring a givenmetabolite. The additional step and capability of synthesizing aninternal standard is required.

Another challenge to the use of metabolite and lipid based biomarkerdetection is the sensitivity of metabolomics to population differences(e.g. population bias). Although metabolite profiles/fingerprints havethe potential to act as indicators of disease, they are also verysensitive to minor differences in the biological background, andtherefore also vary significantly across different populations.Therefore, a challenge in metabolomics based diagnostics is finding amethod that is sensitive to the disease of interest, while at the sametime robust to variations across e.g. different populations.

Likewise, metabolites and lipids are quite sensitive molecules to thepre-analytical treatment of a sample, including sample collectionmethod, storage conditions, and other preparation steps. As a result,measurements of metabolites and lipids are influenced by, e.g.hemolysis, lipemia, sample time at room temperature before serumextraction and freezing, and the freeze-thaw cycles applied to thesamples. Accordingly, sample collection and preparation methods thatcomprise appropriate quality controls and/or yield consistentmeasurements of target biomarkers must be developed and employed.

There is a need for diagnostics to identify the presence, stage, and/orrisk of colorectal cancer and/or adenomatous polyps in a subject.Metabolomics and mass spectrometry based diagnostics is a promisingapproach, but requires identifying an appropriate panel of markers thatare sensitive to the presence of colorectal cancer and/or adenomatouspolyps in a subject, while at the same time robust to populationvariations, such as e.g. gender, age, ethnicity. Additionally,appropriate internal standards, and sample handling and quality controlmethods that enable accurate and reliable measurements are required.

SUMMARY

Described herein are sets of metabolite and lipid (e.g., fatty acid)markers that can be used in the detection of early stage colorectalcancer and/or early development of adenomatous polyps. Presented hereinare illustrative pathology-linked panels. In certain embodiments, themarkers presented herein (or subsets thereof) are used as a panel fordetecting either colorectal cancer or adenomatous polyps at the sametime. The markers presented herein include metabolites and lipids (e.g.,fatty acids) freely detectable and accurately quantifiable in humanserum. In certain embodiments, the sample may be plasma, urine, saliva,whole blood, dried blood spot or dried serum spot.

Also disclosed herein are methods and systems for improved samplehandling and quality control.

In one aspect, the disclosed technology is direct to a methodcomprising: measuring, by mass spectrometry, a level of each of aplurality of species in a biological sample obtained from a humansubject, wherein each of the plurality of species is at least one of ametabolite and a lipid (e.g., a fatty acid) and the plurality of speciescomprises:

and 3Me-glutaryl carnitine

determining a ratio of the measured level of PUFA 446 and the measuredlevel of S192; and determining at least one of a presence of, a stageof, and a risk of colorectal cancer in the human subject based, at leastin part, on the ratio of the measured level of PUFA 446 and the measuredlevel of S192.

In certain embodiments, the plurality of species comprises one or moremembers in addition to PUFA 446 and S192 selected from the groupconsisting of the species listed in Table 12 and Table 13. In certainembodiments, the plurality of species comprises all of the specieslisted in Table 12 and 13 and the method comprises: determining at leastone of a presence of, a stage of, and a risk of colorectal cancer in thehuman subject based, at least in part, on measured values for the ratiosof species listed in Table 12; and determining at least one of apresence of, a stage of, and a risk of adenomatous polyps in the humansubject based, at least in part, on measured values for the ratios ofspecies listed in Table 13.

In certain embodiments, the measuring step comprises measuring the levelof each of the plurality of species using a LC-MS, GC-MS, DESI, or DARTtechnique.

In certain embodiments, the method comprises: determining at least oneof a presence of, a risk of, and a stage of colorectal cancer based, atleast in part, on a ratio of the measured value of a polyunsaturatedfatty acid and the measured value of another of the plurality of speciesbeing lower than a representative ratio for a control population. Incertain embodiments, the polyunsaturated fatty acid is a species listedin FIG. 1.

In certain embodiments, the method comprises: determining at least oneof a presence of, a risk of, and a stage of colorectal cancer based, atleast in part, on a ratio of the measured value of choline (S49) and themeasured value of N1,N12-diacetylsperimne (S236) being lower than arepresentative ratio for a control population.

In certain embodiments, at least one of the plurality of species isselected from the group consisting of:

In certain embodiments, the biological sample comprises serum. Incertain embodiments, the biological sample is serum, plasma, urine,saliva, whole blood, a dried blood spot, or a dried serum spot.

In certain embodiments, the method comprises introducing at least aportion of the biological sample into a C18 50 mm column, a C18 100 mmcolumn, or an amide column to determine a quantification of metabolites,lipids or polar metabolic compounds, respectively, of the plurality ofspecies. In certain embodiments, the method comprises introducing atleast a portion of the biological sample into a mass spectrometer by FIAbased direct infusion injection to measure the level of apolyunsaturated fatty acid (e.g., in a semi-quantitative way). Incertain embodiments, the method comprises measuring a stableisotopically labeled reference standard.

In one aspect, the disclosed technology is directed to a methodcomprising: measuring, by mass spectrometry, a level of each of aplurality of species in a biological sample obtained from a humansubject, wherein each of the plurality of species is at least one of ametabolite and a lipid (e.g., a fatty acid) and the plurality of speciescomprises α-linolenic acid

and L-cysteine S-sulfate

determining a ratio of the measured level of S69 and the measured levelof S153; and determining at least one of a presence of, a stage of, anda risk of adenomatous polyps in the human subject based, at least inpart, on the ratio of the measured level of S69 and the measured levelof S153.

In certain embodiments, the method comprises: determining at least oneof a presence of, a risk of, and a stage of adenomatous polyps based, atleast in part, on a ratio of the measured value of a species in theplurality of species and the measured value of S153 or hippuric acid(S63) being higher than a representative ratio for a control population.In certain embodiments, the species in the plurality of species isoctanoylcarnitine (AC 8:0) (S109), aspartylphenylalanine (S227), or S69.

In certain embodiments, the method comprises: determining at least oneof a presence of, a risk of, and a stage of adenomatous polyps based, atleast in part, on the measured level of S153 being lower than arepresentative level for a control population.

In certain embodiments, the biological sample comprises serum. Incertain embodiments, the biological sample is serum, plasma, urine,saliva, whole blood, a dried blood spot, or a dried serum spot.

In certain embodiments, the method comprises: introducing at least aportion of the biological sample into a C18 50 mm column, a C18 100 mmcolumn, or an amide column to determine a quantification of metabolites,lipids or polar metabolic compounds, respectively, of the plurality ofspecies. In certain embodiments, the method comprises: introducing atleast a portion of the biological sample into a mass spectrometer by FIAbased direct infusion injection to measure the level of apolyunsaturated fatty acid (e.g., in a semi-quantitative way). Incertain embodiments, the method comprises measuring a stableisotopically labeled reference standard.

In certain embodiments, the method comprises comparing certainmetabolite ratios calculated for colorectal cancer and/or adenomatouspolyp patients to a representative ratio for a control population.

In certain embodiments, the biological sample is obtained from the humansubject by collecting blood from the subject when the subject is seatedin an upright position, into evacuated blood collection tubes with noanticoagulant and leaving the blood to clot. In certain embodiments,methods comprise: checking the biological sample for hemolysis and/orlipemia prior to the measuring step; and excluding samples that haveundergone lipemia and/or hemolysis. In certain embodiments, methodscomprise measuring a level of a species in the biological sample that isindicative of a length of time the biological sample was kept at roomtemperature; and determining the length of time the biological samplewas kept at room temperature.

It is contemplated that limitations presented with reference to aparticular aspect of the invention may, in certain embodiments, beapplicable to another aspect of the invention.

Definitions

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration”: As used herein, the term “administration” refers tothe administration of a composition to a subject or system.Administration to an animal subject (e.g., to a human) may be by anyappropriate route. For example, in some embodiments, administration maybe bronchial (including by bronchial instillation), buccal, enteral,interdermal, intra-arterial, intradermal, intragastric, intramedullary,intramuscular, intranasal, intraperitoneal, intrathecal, intravenous,intraventricular, within a specific organ (e. g. intrahepatic), mucosal,nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal(including by intratracheal instillation), transdermal, vaginal andvitreal. In some embodiments, administration may involve intermittentdosing. In some embodiments, administration may involve continuousdosing (e.g., perfusion) for at least a selected period of time. As isknown in the art, antibody therapy is commonly administered parenterally(e.g., by intravenous or subcutaneous injection).

“Biological Sample”: As used herein, the term “biological sample”typically refers to a sample obtained or derived from a biologicalsource (e.g., a tissue or organism or cell culture) of interest, asdescribed herein. In some embodiments, a source of interest comprises anorganism, such as an animal or human. In some embodiments, a biologicalsample is or comprises biological tissue or fluid. In some embodiments,a biological sample may be or comprise bone marrow; blood; blood cells;ascites; tissue or fine needle biopsy samples; cell-containing bodyfluids; free floating nucleic acids; sputum; saliva; urine;cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph;gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasalswabs; washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; feces, other body fluids, secretions,and/or excretions; and/or cells therefrom, etc. In some embodiments, abiological sample is or comprises cells obtained from an individual. Insome embodiments, obtained cells are or include cells from an individualfrom whom the sample is obtained. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc.

“Biomarker”: The term “biomarker” is used herein, consistent with itsuse in the art, to refer to a to an entity whose presence, level, orform, correlates with a particular biological event or state ofinterest, so that it is considered to be a “marker” of that event orstate. To give but a few examples, in some embodiments, a biomarker maybe or comprises a marker for a particular disease state, or forlikelihood that a particular disease, disorder or condition may develop.In some embodiments, a biomarker may be or comprise a marker for aparticular disease or therapeutic outcome, or likelihood thereof. Thus,in some embodiments, a biomarker is predictive, in some embodiments, abiomarker is prognostic, in some embodiments, a biomarker is diagnostic,of the relevant biological event or state of interest. A biomarker maybe an entity of any chemical class. For example, in some embodiments, abiomarker may be or comprise a nucleic acid, a polypeptide, a lipid, acarbohydrate, a small molecule, an inorganic agent (e.g., a metal orion), or a combination thereof. In some embodiments, a biomarker is acell surface marker. In some embodiments, a biomarker is intracellular.In some embodiments, a biomarker is found outside of cells (e.g., issecreted or is otherwise generated or present outside of cells, e.g., ina body fluid such as blood, urine, tears, saliva, cerebrospinal fluid,etc.

“Biomolecule”: As used herein, “biomolecule” refers to bioactive,diagnostic, and prophylactic molecules. Biomolecules that can be used inthe present invention include, but are not limited to, synthetic,recombinant or isolated peptides and proteins such as antibodies andantigens, receptor ligands, enzymes, and adhesion peptides; nucleotidesand polynucleic acids such as DNA and antisense nucleic acid molecule;activated sugars and polysaccharides; bacteria; viruses; and chemicaldrugs such as antibiotics, anti-inflammatories, and antifungal agents.

“Blood component”: As used herein, “blood component” refers to anycomponent of whole blood, including red blood cells, white blood cells,platelets, endothelial cells, mesothelial cells or epithelial cells.Blood components also include the components of plasma, such asproteins, metabolites, lipids, nucleic acids, and carbohydrates, and anyother cells that can be present in blood, due to pregnancy, organtransplant, infection, injury, or disease.

“Cancer”: The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and“carcinoma”, are used interchangeably herein to refer to cells thatexhibit relatively abnormal, uncontrolled, and/or autonomous growth, sothat they exhibit an aberrant growth phenotype characterized by asignificant loss of control of cell proliferation. In general, cells ofinterest for detection or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. The teachings of the present disclosure may berelevant to any and all cancers. To give but a few, non-limitingexamples, in some embodiments, teachings of the present disclosure areapplied to one or more cancers such as, for example, hematopoieticcancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's),myelomas and myeloproliferative disorders; sarcomas, melanomas,adenomas, carcinomas of solid tissue, squamous cell carcinomas of themouth, throat, larynx, and lung, liver cancer, genitourinary cancerssuch as prostate, cervical, bladder, uterine, and endometrial cancer andrenal cell carcinomas, bone cancer, pancreatic cancer, skin cancer,cutaneous or intraocular melanoma, cancer of the endocrine system,cancer of the thyroid gland, cancer of the parathyroid gland, head andneck cancers, breast cancer, gastro-intestinal cancers and nervoussystem cancers, benign lesions such as papillomas, and the like.

“Comparable”: As used herein, the term “comparable” refers to two ormore agents, entities, situations, sets of conditions, etc., that maynot be identical to one another but that are sufficiently similar topermit comparison there between so that conclusions may reasonably bedrawn based on differences or similarities observed. In someembodiments, comparable sets of conditions, circumstances, individuals,or populations are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Thoseof ordinary skill in the art will understand, in context, what degree ofidentity is required in any given circumstance for two or more suchagents, entities, situations, sets of conditions, etc., to be consideredcomparable. For example, those of ordinary skill in the art willappreciate that sets of circumstances, individuals, or populations arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences in results obtained or phenomena observedunder or with different sets of circumstances, individuals, orpopulations are caused by or indicative of the variation in thosefeatures that are varied.

“Diagnostic information”: As used herein, “diagnostic information” or“information for use in diagnosis” is information that is useful indetermining whether a patient has a disease, disorder or conditionand/or in classifying a disease, disorder or condition into a phenotypiccategory or any category having significance with regard to prognosis ofa disease, disorder or condition, or likely response to treatment(either treatment in general or any particular treatment) of a disease,disorder or condition. Similarly, “diagnosis” refers to providing anytype of diagnostic information, including, but not limited to, whether asubject is likely to have or develop a disease, disorder or condition,state, staging or characteristic of a disease, disorder or condition asmanifested in the subject, information related to the nature orclassification of a tumor, information related to prognosis and/orinformation useful in selecting an appropriate treatment. Selection oftreatment may include the choice of a particular therapeutic agent orother treatment modality such as surgery, radiation, etc., a choiceabout whether to withhold or deliver therapy, a choice relating todosing regimen (e.g., frequency or level of one or more doses of aparticular therapeutic agent or combination of therapeutic agents), etc.

“Marker”: A marker, as used herein, refers to an entity or moiety whosepresence or level is a characteristic of a particular state or event. Insome embodiments, presence or level of a particular marker may becharacteristic of presence or stage of a disease, disorder, orcondition. To give but one example, in some embodiments, the term refersto a gene expression product that is characteristic of a particulartumor, tumor subclass, stage of tumor, etc. Alternatively oradditionally, in some embodiments, a presence or level of a particularmarker correlates with activity (or activity level) of a particularsignaling pathway, for example that may be characteristic of aparticular class of tumors. The statistical significance of the presenceor absence of a marker may vary depending upon the particular marker. Insome embodiments, detection of a marker is highly specific in that itreflects a high probability that the tumor is of a particular subclass.Such specificity may come at the cost of sensitivity (i.e., a negativeresult may occur even if the tumor is a tumor that would be expected toexpress the marker). Conversely, markers with a high degree ofsensitivity may be less specific that those with lower sensitivity.According to the present invention a useful marker need not distinguishtumors of a particular subclass with 100% accuracy. A marker may be ametabolite, lipid, fatty acid, and/or polyunsaturated fatty acid. Incertain embodiments, the term marker may refer to a ratio of twoentities (e.g., moieties).

“Prevent or prevention”: as used herein when used in connection with theoccurrence of a disease, disorder, and/or condition, refers to reducingthe risk of developing the disease, disorder and/or condition and/or todelaying onset of one or more characteristics or symptoms of thedisease, disorder or condition. Prevention may be considered completewhen onset of a disease, disorder or condition has been delayed for apredefined period of time.

“Prognostic and predictive information”: As used herein, the terms“prognostic information” and “predictive information” are used to referto any information that may be used to indicate any aspect of the courseof a disease or condition either in the absence or presence oftreatment. Such information may include, but is not limited to, theaverage life expectancy of a patient, the likelihood that a patient willsurvive for a given amount of time (e.g., 6 months, 1 year, 5 years,etc.), the likelihood that a patient will be cured of a disease, thelikelihood that a patient's disease will respond to a particular therapy(wherein response may be defined in any of a variety of ways).Prognostic and predictive information are included within the broadcategory of diagnostic information.

“Prevention”: The term “prevention”, as used herein, refers to a delayof onset, and/or reduction in frequency and/or severity of one or moresymptoms of a particular disease, disorder or condition. In someembodiments, prevention is assessed on a population basis such that anagent is considered to “prevent” a particular disease, disorder orcondition if a statistically significant decrease in the development,frequency, and/or intensity of one or more symptoms of the disease,disorder or condition is observed in a population susceptible to thedisease, disorder, or condition. Prevention may be considered completewhen onset of a disease, disorder or condition has been delayed for apredefined period of time.

“Ratio”: As used herein, the term “ratio” refers to a calculablerelationship used to compare amounts of two species that indicates therelative amounts of the species. The species may be markers, such asmetabolites and/or lipids (e.g., fatty acids), for example. A ratio maybe a direct proportion or inverse proportion (e.g., a first amountdivided by a second amount or the second amount divided by the firstamount, respectively). A ratio may be weighted and/or normalized (eitherthe numerator, the denominator, or both). The two amounts may bephysical quantities or arbitrary values that correspond to physicalquantities. For example, a ratio may be calculated from two intensityamounts (i.e., in arbitrary units) in two species (e.g., markers)measured by a mass spectrometry technique.

“Reference”: As used herein, the term “reference” describes a standardor control relative to which a comparison is performed. For example, insome embodiments, an agent, animal, individual, population, sample,sequence or value of interest is compared with a reference or controlagent, animal, individual, population, sample, sequence or value. Insome embodiments, a reference or control is tested and/or determinedsubstantially simultaneously with the testing or determination ofinterest. In some embodiments, a reference or control is a historicalreference or control, optionally embodied in a tangible medium.Typically, as would be understood by those skilled in the art, areference or control is determined or characterized under comparableconditions or circumstances to those under assessment. Those skilled inthe art will appreciate when sufficient similarities are present tojustify reliance on and/or comparison to a particular possible referenceor control.

“Risk”: as will be understood from context, “risk” of a disease,disorder, and/or condition comprises likelihood that a particularindividual will develop a disease, disorder, and/or condition (e.g., aradiation injury). In some embodiments, risk is expressed as apercentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodimentsrisk is expressed as a risk relative to a risk associated with areference sample or group of reference samples. In some embodiments, areference sample or group of reference samples have a known risk of adisease, disorder, condition and/or event (e.g., a radiation injury). Insome embodiments a reference sample or group of reference samples arefrom individuals comparable to a particular individual. In someembodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

“Sample”: As used herein, the term “sample” typically refers to abiological sample obtained or derived from a source of interest, asdescribed herein. In some embodiments, a source of interest comprises anorganism, such as an animal or human. In some embodiments, a biologicalsample is or comprises biological tissue or fluid. In some embodiments,a biological sample may be or comprise bone marrow; blood; blood cells;ascites; tissue or fine needle biopsy samples; cell-containing bodyfluids; free floating nucleic acids; sputum; saliva; urine;cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph;gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasalswabs; washings or lavages such as a ductal lavages or broncheoalveolarlavages; aspirates; scrapings; bone marrow specimens; tissue biopsyspecimens; surgical specimens; feces, other body fluids, secretions,and/or excretions; and/or cells therefrom, etc. In some embodiments, abiological sample is or comprises cells obtained from an individual. Insome embodiments, obtained cells are or include cells from an individualfrom whom the sample is obtained. In some embodiments, a sample is a“primary sample” obtained directly from a source of interest by anyappropriate means. For example, in some embodiments, a primarybiological sample is obtained by methods selected from the groupconsisting of biopsy (e.g., fine needle aspiration or tissue biopsy),surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.In some embodiments, as will be clear from context, the term “sample”refers to a preparation that is obtained by processing (e.g., byremoving one or more components of and/or by adding one or more agentsto) a primary sample. For example, filtering using a semi-permeablemembrane. Such a “processed sample” may comprise, for example nucleicacids or proteins extracted from a sample or obtained by subjecting aprimary sample to techniques such as amplification or reversetranscription of mRNA, isolation and/or purification of certaincomponents, etc.

“Small molecule”: As used herein, the term “small molecule” means a lowmolecular weight organic and/or inorganic compound. In general, a “smallmolecule” is a molecule that is less than about 5 kilo-Daltons (kD) insize. In some embodiments, a small molecule is less than about 4 kD, 3kD, about 2 kD, or about 1 kD. In some embodiments, the small moleculeis less than about 800 Daltons (D), about 600 D, about 500 D, about 400D, about 300 D, about 200 D, or about 100 D. In some embodiments, asmall molecule is less than about 2000 g/mol, less than about 1500g/mol, less than about 1000 g/mol, less than about 800 g/mol, or lessthan about 500 g/mol. In some embodiments, a small molecule is not apolymer. In some embodiments, a small molecule does not include apolymeric moiety. In some embodiments, a small molecule is not a proteinor polypeptide (e.g., is not an oligopeptide or peptide). In someembodiments, a small molecule is not a polynucleotide (e.g., is not anoligonucleotide). In some embodiments, a small molecule is not apolysaccharide. In some embodiments, a small molecule does not comprisea polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid,etc.). In some embodiments, a small molecule is not a lipid. In someembodiments, a small molecule is a modulating agent. In someembodiments, a small molecule is biologically active. In someembodiments, a small molecule is detectable (e.g., comprises at leastone detectable moiety). In some embodiments, a small molecule is atherapeutic. Those of ordinary skill in the art, reading the presentdisclosure, will appreciate that certain small molecule compoundsdescribed herein may be provided and/or utilized in any of a variety offorms such as, for example, salt forms, protected forms, pro-drug forms,ester forms, isomeric forms (e.g., optical and/or structural isomers),isotopic forms, etc. In some embodiments, reference to a particularcompound may relate to a specific form of that compound. In someembodiments, reference to a particular compound may relate to thatcompound in any form. In some embodiments, where a compound is one thatexists or is found in nature, that compound may be provided and/orutilized in accordance in the present invention in a form different fromthat in which it exists or is found in nature. Those of ordinary skillin the art will appreciate that a compound preparation including adifferent level, amount, or ratio of one or more individual forms than areference preparation or source (e.g., a natural source) of the compoundmay be considered to be a different form of the compound as describedherein. Thus, in some embodiments, for example, a preparation of asingle stereoisomer of a compound may be considered to be a differentform of the compound than a racemic mixture of the compound; aparticular salt of a compound may be considered to be a different formfrom another salt form of the compound; a preparation containing oneconformational isomer ((Z) or (E)) of a double bond may be considered tobe a different form from one containing the other conformational isomer((E) or (Z)) of the double bond; a preparation in which one or moreatoms is a different isotope than is present in a reference preparationmay be considered to be a different form; etc.

“Subject”: As used herein, the term “subject” includes humans andmammals (e.g., mice, rats, pigs, cats, dogs, and horses). In manyembodiments, subjects are be mammals, particularly primates, especiallyhumans. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; poultry such as chickens,ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. In some embodiments (e.g.,particularly in research contexts) subject mammals will be, for example,rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine suchas inbred pigs and the like.

“Substantially”: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

“Susceptible to”: An individual who is “susceptible to” a disease,disorder, or condition (e.g., influenza) is at risk for developing thedisease, disorder, or condition. In some embodiments, an individual whois susceptible to a disease, disorder, or condition does not display anysymptoms of the disease, disorder, or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, or condition hasnot been diagnosed with the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder, orcondition is an individual who has been exposed to conditions associatedwith development of the disease, disorder, or condition. In someembodiments, a risk of developing a disease, disorder, and/or conditionis a population-based risk (e.g., family members of individualssuffering from the disease, disorder, or condition).

“Treatment”: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition. Such treatment can be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment can beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment canbe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment canbe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

“Early stage”: As used herein, the term “early stage” refers to alocalized stage where cancer has not yet spread to nearby lymph nodes(NO) or to distant sites (MO). For example, pathologically it would becancer stages from stage 0 to stage II C.

Drawings are presented herein for illustration purposes, not forlimitation.

BRIEF DESCRIPTION OF THE DRAWINGS

Drawings are presented herein for illustration purposes, not forlimitation. The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee. The foregoing and otherobjects, aspects, features, and advantages of the invention will becomemore apparent and may be better understood by referring to the followingdescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 shows the chemical structure of 6 PUFA (polyunsaturated fattyacid) molecules;

FIG. 2 shows chemical structures of α-linolenic acid, 3Me-GlutarylCarnitine, L-cysteine S-Sulfate;

FIG. 3 shows a set of qualitative indicators that may be used for visualcomparison to qualitative determine hemolysis in serum samples,according to an illustrative embodiment of the present invention;

FIG. 4 shows a bias test performed on marker PUFA 468, wherein panel Arepresents an age versus concentration plot for control samples, panel Brepresents an age versus concentration plot for colorectal cancerpatients, panel C represents an age versus concentration plot foradenomatous polyp patients, panel D represents box-plots for malepatients, and panel E represents box-plots for female patients;

FIG. 5 shows a linear increase obtained with increase hemolysis forA414-385 values represented in Table 5, indicating good accuracy forusing A414-385 as measurement on samples displaying only hemolysis;

FIG. 6 shows a linear relationship between the extent of lipemia (interms of A385 values, x axis) and A A414-A385 estimation (y axis) with aresulting quantitative relationship derived from a linear regressionalso shown;

FIG. 7 shows the sensitivity and specificity of a marker panel based onthe list of markers in Table 9;

FIG. 8 shows the sensitivity of a marker panel in detecting thedifferent stages of CRC based on the list of markers in Table 9;

FIG. 9 shows an example of 4 individual features (markers or ratios oftwo markers) that have high significance for detecting colorectal cancer(the markers are selected from the subset listed in Table 9); and

FIG. 10 shows an example of 4 individual features (markers or ratios oftwo markers) that have high significance for detecting adenomatouspolyps (the markers are selected from the subset listed in Table 9).

DETAILED DESCRIPTION

Throughout the description, where compositions are described as having,including, or comprising specific components, or where methods aredescribed as having, including, or comprising specific steps, it iscontemplated that, additionally, there are compositions of the presentinvention that consist essentially of, or consist of, the recitedcomponents, and that there are methods according to the presentinvention that consist essentially of, or consist of, the recitedprocessing steps.

It should be understood that the order of steps or order for performingcertain action is immaterial so long as the invention remains operable.Moreover, two or more steps or actions can be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim. Headers are providedfor the convenience of the reader and are not intended to be limitingwith respect to the claimed subject matter.

Marker Panel, and Classification Approach

Described herein are sets of metabolite and lipid markers that can beused as a full panel or as a subset of markers for early stagecolorectal cancer and early development of adenomatous polyp detection.In certain embodiments, these markers (or subsets thereof) are separatedinto pathology-linked panels. In certain embodiments, these markers (orsubsets thereof) are used as a single panel for detecting bothcolorectal cancer and adenomatous polyps at the same time. In certainembodiments, these markers are metabolites and lipids (e.g., fattyacids) freely detectable and accurately quantifiable in human serum. Incertain embodiments, the sample may be plasma, urine, saliva, wholeblood, dried blood spot or dried serum spot.

In certain embodiments, markers are measured using mass spectrometry. Insome embodiments, markers are measured using column-basedliquid-chromatography mass spectrometry (LC-MS). For example, a C18 50mm column may be used for metabolite quantification (e.g. using an ABSciex TQ mass spectrometer). Alternatively, an Amide column may be usedfor detecting polar metabolic compounds (e.g. using an AB Sciex TQ massspectrometer). For example, an ACQUITY UPLC BEH C18 Column, 130 Å, 1.7μm, 2.1 mm×50 mm may be used for metabolite quantification.Alternatively, an ACQUITY UPLC BEH Amide Column, 130 Å, 1.7 μm, 2.1mm×150 mm may be used for detecting polar metabolic compounds. A C18 100mm column may be used for lipid quantification (e.g. using an AB SciexQTRAP). In some embodiments, detection of species uses direct injectionmethods, for example, FIA based direct injection for analyzing fattyacids (e.g. polyunsaturated fatty acids (PUFA)) (e.g. using an AB SciexQTRAP).

The panel of markers shown in Table 1 and Table 2 was identified usingan exemplary assay development process described herein. Table 1 liststhe names of the different markers, along with the detection method usedto measure each marker (“Column” heading in the table). Certain markersmay be measured by more than one different method. Chemical structuresof several of the molecules are shown in FIG. 1 and FIG. 2. In certainembodiments, the molecules represented in FIG. 1 and FIG. 2 are used asor in markers in a panel for diagnosing colorectal cancer and/oradenomatous polyps. FIG. 1 Referring now to FIG. 1, the sixpolyunsaturated fatty acids represented in the figure are labeledaccording to their molecular weight for ease in distinguishing betweenspecies and (e.g., “PUFA 446” corresponds to the polyunsaturated fattyacid represented in FIG. 1 having molecular weight of 446 g/mol).

TABLE 1 Listing of species identified Lab code Name Column1-(1-enyl-stearoyl)-GPE (P-18:0)* Amide S261 Alanine Amide S49 CHOLINEAmide S325 glycerophosphoethanolamine (GPE-2) Amide S153 L-cysteineS-sulfate Amide S110 L-Histidine Amide S125 L-Lysine Amide S132L-proline Amide S236 N1,N12-Diacetylspermine Amide S111NG,NG-dimethylarginine (asym) (ADMA) Amide S78 NICOTINAMIDE Amide S179sn-Glycero-3-phosphocholine Amide S175 1,18-Octadecanedicarboxylic acidC18 S3 14:0 Lyso PC C18 S15 16:0 Lyso PC C18 S103 1-Methyladenosine C18S105 1-O-Palmityl-sn-glycero-3-phosphocholine C18 S1932-D-Mannopyranosyl-L-tryptophan C18 S305 3-(3-hydroxyphenyl)propionateC18 S192 3Me-Glutaryl Carnitine C18 S285 Acetoacetate C18 S227Aspartylphenylalanine C18 S295 Azelaic acid C18 S168 Delta-ValerolactamC18 S62 GLYCOCHOLIC ACID C18 S52 GLYCODEOXYCHOLATE C18 S63 HIPPURIC ACIDC18 S65 KYNURENIC ACID C18 S69 α-LINOLENIC ACID C18 S150 L-Pyrogutamicacid C18 S1 L-Tryptophan C18 S100 L-tyrosine C18 S133 Lyso PC 18:0 C18S126 Lyso PC 20:0 C18 S171 N-(2-Furoyl)glycine C18 S166 N-AcetylcytidineC18 S76 N-ACETYLGLYCINE C18 S170 Nonanoic Acid C18 S109Octanoylcarnitine (AC 8:0) C18 S147 Propionyl L-carnitine (AC 3:0) C18S10 Sebacic acid C18 S94 XANTHUNERIC ACID C18 S176 y-cehc C18 PUFA6 PUFA468 C18-100 mm AC 4:0 C18-100 mm AC 14:0 C18-100 mm AC 16:0 C18-100 mmLPA 16:0 C18-100 mm LPC 18:2 C18-100 mm LPC 20:3 C18-100 mm LPC O-16:0C18-100 mm LPE 18:2 C18-100 mm LPI 18:0 C18-100 mm LPI 18:1 C18-100 mmLPI 20:3 C18-100 mm PC 34:2 (16:1_18:1) C18-100 mm PC 36:1 C18-100 mm PC36:1 (18:1_18:0) C18-100 mm PC 36:4 (18:2/18:2) C18-100 mm PC 36:4(16:0/20:4) C18-100 mm PC 38:5 C18-100 mm PC 38:5 (16:0/22:5) C18-100 mmPC 38:5 (18:2/20:3) C18-100 mm PC 38:6 (18:2/20:4) C18-100 mm PC 40:4(18:0/22:4) C18-100 mm PC O-34:2 (16:0/18:2) C18-100 mm PC O-34:3C18-100 mm PC O-36:3 C18-100 mm PC O-36:4 (16:0/20:4) C18-100 mm PCO-38:4 C18-100 mm PC O-40:1 C18-100 mm PC O-42:1 C18-100 mm PC O-44:4C18-100 mm PI 36:3 (18:1/18:2) C18-100 mm PI 36:1 (18:0/18:1) C18-100 mmPI 36:2 (18:1/18:1) C18-100 mm S18 S1P (Spingosine 1-P) C18-100 mm SM38:0 C18-100 mm PUFA1 PUFA 446 FIA PUFA2 PUFA 448 FIA PUFA3 PUFA 450 FIAPUFA4 PUFA 464 FIA PUFA5 PUFA 466 FIA PUFA6 PUFA 468 FIA

TABLE 2 Listing of markers, along with indications of which markers maybe interchanged with each other, or used as quality controls. Severalmarkers in addition to those listed in Table 1 are also included UDXMethod of Code Name of the Molecule Detection Comments1-(1-enyl-stearoyl)-GPE (P-18:0)* Amide Putative identification S261Alanine Amide S49 CHOLINE Amide S325 Glycerophosphoethanolamine (GPE-2)Amide S153 L-cysteine S-Sulfate Amide S110 L-Histidine Amide Can beinterchanged with S236 S125 L-Lysine Amide S132 L-proline Amide S236N1,N12-Diacetylspermine Amide S111 NG,NG-dimethylarginine (asym) (ADMA)Amide S78 NICOTINAMIDE Amide S179 Sn-Glycero-3-phosphocholine Amide S1751,18-Octadecanedicarboxylic acid C18 S3 14:0 Lyso PC C18 Can beinterchanged with S15, S126, S133, LPC 20:3 S15 16:0 Lyso PC C18 Can beinterchanged with S3, S126, S133 S103 1-Methyladenosine C18 Can beinterchanged with S166 S105 1-O-Palmityl-sn-glycero-3-phosphocholine C18S193 2-D-Mannopyranosyl-L-tryptophan C18 S3053-(3-hydroxyphenyl)propionate C18 S192 3Me-Glutaryl Carnitine C18 S285Acetoacetate C18 S227 Aspartylphenylalanine C18 S295 Azelaic acid C18Too low concentration (may be excluded from the panel entirely) S168Delta-Valerolactam C18 Can be interchanged with S176 S61GLYCOCHENODEOXYCHOLATE C18 Marker used for evaluating icterus S62GLYCOCHOLIC ACID C18 Marker used for evaluating icterus S52GLYCODEOXYCHOLATE C18 Marker used for evaluating icterus S63 HIPPURICACID C18 S65 KYNURENIC ACID C18 Linked to S1 and S94 S69 LINOLENIC ACIDC18 S150 L-Pyroqutamic acid C18 Marker indicating time at roomtemperature S1 L-Tryptophan C18 Can be interchanged with S94 S100L-tyrosine C18 S133 Lyso PC 18:0 C18 Can be interchanged with S3, S126,S133 S126 Lyso PC 20:0 C18 Can be interchanged with S3, S126, S133 S171N-(2-Furoyl)glycine C18 Can be interchanged with S170 S166N-Acetylcytidine C18 Can be interchanged with S103 S76 N-ACETYLGLYCINEC18 S170 Nonanoic Acid C18 Can be interchanged with S171 S109Octanoylcarnitine (AC 8:0) C18 S147 Propionyl L-carnitine (AC 3:0) C18S10 Sebacic acid C18 Marker indicating improper sample collection S94XANTHUNERIC ACID C18 S176 y-cehc C18 Can be interchanged with S168 S313N2,N2-dimethylguanosine C18 S321 DSGEGDFXAEGGGVR * (Androsterone C18sulfate S333 N2-methylguanosine C18 PUFA468 C18-100 mm S245 AC 4:0C18-100 mm LPA 16:0 C18-100 mm LPC 18:2 C18-100 mm Can be interchangedwith LPE 18:2 LPC 20:3 C18-100 mm Can be interchanged with S3 LPC O-16:0C18-100 mm Can be interchanged with S3 LPE 18:2 C18-100 mm Can beinterchanged with LPC 18:2 S341 LPI 18:0 C18-100 mm Can be interchangedwith LPI 18:1 LPI 18:1 C18-100 mm LPI 20:3 C18-100 mm PC 34:2(16:1_18:1) C18-100 mm PC 36:1 (18:1/18:0) C18-100 mm PC 36:1(18:1_18:0) C18-100 mm S338 PC 36:4 (18:2/18:2) C18-100 mm Can beinterchanged with PC 38:6 (18:2/20:4) S339 PC 36:4 (16:0/20:4) C18-100mm PC 38:5 (20:4/18:1) C18-100 mm PC 38:5 (16:0/22:5) C18-100 mm PC 38:5(18:2/20:3) C18-100 mm PC 38:6 (18:2/20:4) C18-100 mm Can beinterchanged with PC 36:4 (18:2/18:2) PC 40:4 (18:0/22:4) C18-100 mm PCO-34:2 (16:0/18:2) C18-100 mm Can be interchanged with PC O-36:3(18:1/18:2) PC O-34:3 C18-100 mm PC O-36:3 (18:1/18:2) C18-100 mm Can beinterchanged with PC O-34:2 (16:0/18:2) PC O-36:4 (16:0/20:4) C18-100 mmPC O-38:4 (18:0/20:4) C18-100 mm PC O-40:1 C18-100 mm PC O-42:1 C18-100mm PC O-44:4 C18-100 mm PI 36:3 (18:1)/18:2) C18-100 mm PI 36:1(18:0/18:1) C18-100 mm Can be interchanged with S329, PI 36:3(18:1/18:2) S329 PI 36:2 (18:1/18:1) C18-100 mm Can be interchanged withPI 36:1 (18:0/18:1), PI 36:3 (18:1/18:2) S18 S1P (Spingosine 1-P)C18-100 mm SM 38:0 C18-100 mm S2 Oleoyl L-Carnitine C18-100 mm Can beinterchanged with S127, S135 LPC 15:0 C18-100 mm Can be interchangedwith LPC 18:1, LPC 20:2 LPC 18:1 C18-100 mm Can be interchanged with LPC15:0, LPC 18:1 LPC 20:2 C18-100 mm Can be interchanged with LPC 15:0,LPC 18:1 PC 42:8 C18-100 mm FFA 22:0 C18-100 mm PE(P-18:1/18:1) C18-100mm PE(18:2/18:2) C18-100 mm S342 PC 38:6 (16:0/22:6) C18-100 mm S135(±)-Myristoylcarnitine (AC 14:0) C18-100 mm Can be interchanged withS127, S2 S127 Palmitoyl-L-carnitine (AC 16:0) C18-100 mm Can beinterchanged with S2, S135 PUFA 446 FIA Can be interchanged with PUFA468 PUFA 448 FIA PUFA 450 FIA PUFA 464 FIA PUFA 466 FIA PUFA 468 FIA Canbe interchanged with PUFA 446 LFA 538 FIA Putative identification LFA592 FIA Putative identification LFA 594 FIA Putative identification

In certain embodiments, measurements of all or small subsets of (e.g.,at least 2, at least 3, at least 4, at least 5, between 2 and 80,between 2 and 50, between 3 and 50, between 4 and 50, between 10 and 40,no greater than 80, no greater than 70, no greater than 60, no greaterthan 50, no greater than 40, no greater than 30, no greater than 20,e.g., 2, e.g., 3, e.g., 4, e.g., 10, e.g., 12, e.g. 16, e.g., 30 of)these markers may be used in a predictive model (e.g. based onstatistical pattern recognition methods, such as, e.g. Naïve Bayesclassifiers, Support Vector Machines (SVM), Random Forests (RF)) todistinguish between healthy and diseased states related to colorectalcancer. In certain other embodiments, an individual marker may be usedin the predictive model. In certain embodiments, a patient or sample maybe identified (e.g. classified) as having colorectal cancer oradenomatous polyp or either colorectal cancer or adenomatous polyp usingthe predictive model.

The development of a predictive model, or classifier, may follow thegeneral two-step approach of (1) training, followed by (2)classification, or testing. The training step is used to build thepredictive model using data (e.g. measurements of markers) thatcorrespond to samples that are known to belong to specified classes, andcreating a classifier on the basis of that known content that accuratelyidentifies the class (e.g. positive or negative for colorectal cancer,adenomatous polyp) based on the values of measurements of a set ofmarkers from the panel. As would be appreciated by one of skill in theart, this step may comprise a feature selection process, wherein thebest markers are identified (e.g. an optimal set of a predefined numberof markers). To find thresholds for given content, one needs to trainthe classifier with sample content that represents members of all of theclasses. Training may be carried out on a portion (e.g. 70%) of thedata.

In certain embodiments, the remaining portion of the data (e.g. 30%) maybe used to test the classifier, by using the model to predict the healthstate of patients in the testing set. Since the real health status ofeach individual in the testing set is known, the accuracy of the modelcan be assessed by comparing the real classes with predicted classes.

This approach can be used to assess the performance of the classifier,by calculating e.g. true and false positive rates, as well as e.g.sensitivities (the true positive rate) and specificity (1—the falsepositive rate) based on particular cut of points for different variableparameters (e.g. cut-off thresholds for particular markers). Varyingparameters may be used to generate standard Receiver OperatingCharacteristic (ROC) curves, which may plot the the true positive rate(TPR) against the false positive rate (FPR) at various thresholdsettings. The area under the ROC curve (AUC) is a measure of how well aparameter can distinguish between two diagnostic groups (e.g. diseaseversus normal).

Clinical predictive algorithms may also be adapted in such a way so thatthe mistake of classifying cancer patients as normal (false negative) isless likely than the mistake of classifying a healthy person as havingcancer (false positive), or, vice versa—e.g., such that the mistake of afalse positive is less likely than the mistake of a false negative.

Certain markers may be measured by multiple methods, such that eachmeasurement may be used as an input to the predictive model (e.g. eachmeasurement of a given species acts as a separate biomarker). Forexample, measuring PUFA 468 using a lipid quantification approach (e.g.using C18-100 mm column) as well as in FIA method has been found toadding additional statistical value to the panel.

Additionally, certain markers may be interchanged with each other (e.g.as a result of being highly correlated). For example, a predictive modelthat uses S3 (14:0 Lyso PC) as an input would perform similarly to onethat uses S15, S126, or S133 in place of S3, but is otherwise identical.Table 2 lists which markers can be interchanged with others.

Certain markers may also be used in quality control measures, forexample as indicators of sample handling and storage conditions as willbe discussed herein. Table 2 also provides indications of these markers.

Several of the markers in Table 1 and Table 2 can be identified asbelong to particular classes of molecules. The predictive value thesemarkers provide for detecting colorectal cancer or polyps is thereforeindicative of the potential predictive value of other moleculesbelonging to the same class. For example, the PUFA fatty acid group isespecially indicative of colorectal cancer. The chemical structures of 6relevant PUFAs are shown in FIG. 1. The drop in the concentration ofthese fatty acids is a clear indicator of strong risk of havingcolorectal cancer.

In certain embodiments, fatty acids, including the PUFA molecules inFIG. 1, represent an important category of markers for use in adiagnostic panel.

Markers related to phenylalanine may also represent an important classof markers.

Additionally, lipids from lysophospholipid class (LPC 14:0, LPC 16:0,LPC 18:2, LPC 20:0 and LPC 20:3) and phosphocholine class (PC 34:2(16:1_18:1), PC 36:1, PC 36:1 (18:1_18:0), PC 36:4 (18:2/18:2), PC 36:4(16:0/20:4), PC 38:5, PC 38:5 (16:0/22:5), PC 38:5 (18:2/20:3), PC 38:6(18:2/20:4), PC 40:4 (18:0/22:4)) are especially indicative ofcolorectal cancer.

Triacylglycerides, phosphoglycerides (e.g. PC, PE, and PI moleculeslisted in Table 1), sterols, and sphingolipids also represent animportant class of relevant biomarkers.

Another important class of molecules comprises acylcartinites.

The carnitines, e.g. S147, AC 4:0, AC 14:0, AC 16:0, S192 and S109, arean important group of molecules especially for detecting adenomas.

Constituent amino acids of elastin, such as proline, leucine, valine andglycine may correspond to an important group of molecules for cancerdetection.

In one embodiment, subsets of the markers identified in Table 1 andTable 2 were identified as providing distinguishable specific signaturesthat would work for 2 different classification problems. These are (i)colorectal cancer vs control, (ii) adenomatous polyp vs control. Forboth of (i)-(ii), a distinct set of markers to be measured as inputs toa predictive model was identified.

Check for Lack of Population Bias Experiment

Because of the challenge population bias presents in metabolomics,markers were checked for robustness to variations in age, gender andethnicity. Markers that have tendency to be highly indicative of age orgender nor ethnicity may introduce bias into analysis algorithm and thuscould potentially result with clinically invalid assay. Accordingly, inone embodiment, only markers that were robust to variations in age,gender and ethnicity were included.

FIG. 4 shows results of an example of a test made on checking for ageand gender bias for the PUFA 468 marker. Panels A-C show concentrationvalues (given in arbitrary units) plotted against the age of thepatients for control patients (panel A), patients with colorectal cancer(panel B), and patients with adenomatous polyps (panel C). A linearregression was used to determine the correlation between age andconcentration by evaluating the r² coefficient. An r² value close to 1is indicative of a strong correlation, while an r² coefficient close to0 indicates no correlation. The highest observed r² value for the datain FIG. 4 is 0.11, which, without wishing to be bound by any theory,means that no age bias can be detected for the data presented in panelsA-C. Panels D-E show box-plots of concentration values or male patients(panel D) and female patients (panel E). Both data sets present similarbehavior of the PUFA 468 marker (e.g. a reduced concentration of thePUFA 468 molecule in patients having CRC), indicating a lack of genderbias.

Similar graphs have been generated for all markers. Markers with stronginclination to having bias were eliminated from consideration.

While the markers themselves do not have biological bias, it is stillpossible to include parameters such as age and gender as importantparameters in a prediction algorithm as physiological markers.

Sample Preparation, Quality Control and Measurement Methods

In addition to population bias, as discussed herein, pre-analytical biasis another important consideration in metabolomics based diagnostics.

In order to address the challenge of pre-analytical bias, strict samplecollection protocols and quality control methods may be employed.

In certain embodiments, sample handling methods that result inhemolysis, protein aggregation or contamination, should be avoided. Forexample, strong mechanical treatment that would cause red cell ruptureshould be avoided.

Exemplary Serum Sample Collection Protocol

The following is an exemplary serum sample collection protocol.

The following are provided for use in sample collection: VACUETTE® SerumClot Activator Tubes with gel separator (red cap, yellow ring) (suppliedby Greiner Bio-One); Matrix 1 mL 2D tube+cap, 1D side racked ST(supplied by Thermoscientific); and a thermometer/hygrometer.

The following equipment and supplies are required at the collectionsite: a pipette and disposable filter tips for serum handling (500 μL,into every matrix2D tubes); a refrigerated centrifuge capable ofchilling to 4° C. and centrifuging at 2000×g; a refrigerated centrifugecapable of chilling to 4° C. and centrifuging at 16000×g; and anultralow freezer capable of chilling to −80° C. (−112° F.).

First, an 8 mL of whole blood should be collected using provided tubesfor serum separation. The tube should be inverted 5-10 times immediatelyafter collection. After collecting 8 mL of whole blood using VACUETTESerum Clot Activator Tubes and inverting, the blood should be leftclotting for half an hour to 1 hour at room temperature. The tubesshould be kept vertical while clotting. The temperature and humidityconditions should be registered during clotting using the providedthermometer/hygrometer.

After the clotting period, the blood should be centrifuged at 2000×g for10 minutes at 4° C. (39.2° F.). The time at which sample is centrifugedshould be registered. Next, a pipette with disposable filter tips isused to transfer serum supernatant obtained via centrifugation intoprovided Matrix2D tubes, divided into aliquots with 500 μL of serum ineach. Great care needs to be taken when aliquoting the samples to notdisturb the red blood cell pellet that forms during centrifugation. Thelast aliquot man have less than 500 μL. A different tip must be used foreach patient and the pipette must be kept in a vertical position duringthe process in order to ensure accurate dispensing volume.

The hemolysis level can be visually inspected during the serum samplecollection process to ensure the collection of viable samples. Visiblyhemolyzed samples are not acceptable for metabolomics studies and shouldbe excluded. FIG. 3 shows a qualitative visual comparison guide that canbe used to determine whether hemolysis has occurred in samples. Theindicators inside the box in FIG. 3 (i.e., the two leftmost indicators)correspond to samples which are valid for use in further analysis. Allaliquots should have similar coloring in order to ensure accurate andconsistent analysis results. In certain embodiments, collected samplescan be stored at room temperature for up to two weeks after collection.In certain embodiments, samples should be shipped at room temperature.

In order to check for adherence appropriate protocols in a clinicalsetting, quality control methods, which are disclosed herein, may beemployed. Quality control methods may be used to check for hemolysis,lipemia and sample storage conditions (e.g. whether the samples hasremained at room temperature too long before serum extraction andstorage). Particular quality control methods have been developed and aredisclosed herein below. Included are methods for measuring hemolysis,lipemia, bilirubin contamination, contamination from certain storagevials, and time at room temperature before serum extraction.

Hemolysis and Lipemia Quality Control Measurements

Hemolysis (or hemolysis), from the Latin hemo (blood) and lysis (tobreak open), is the release of hemoglobin and other intracellularcomponents from erythrocytes to the surrounding plasma, following damageor disruption of the cell membrane.

Lipemic plasma has large lipid particles that include lipoproteins andchylomicrons. As a result, these samples have increased sample turbidityand may result in the prolongation of coagulation results. Interferenceis variable among analyzers.

In certain embodiments, an increase in an optical absorbance measurement(e.g. measured via a NanoDrop® spectrophotometer) at wavelength of A414is correlated with an increase in free hemoglobin concentration. Incertain embodiments, measurements at a wavelength of A385 are indicativeof lipemia. As used herein, the letter “A” preceding a number (e.g., asin “A385”) refers to a wavelength of light where an absorbance peak ismeasured, wherein the number is the wavelength as expressed innanometers (e.g., “measurements at A385” refers to measuring anabsorbance peaks that occurs at a wavelength of 385 nm).

When both lipemia and hemolysis are present in the sample, measurementsat A414 can be affected by presence of lipemia and thus cannot be takenas reliable measurements for evaluating hemolysis. Additionally,measurements at A660-700 may provide an alternative option to be usedfor characterizing lipemia (e.g. may be measured a with Roche Cobas®6000 analyzer).

Since measurements of UV-Vis absorbance at individual wavelengths can beinfluenced by the presence of hemolysis and lipemia, metrics that canprovide reliable indication of hemolysis, but are at the same timerobust to variations in lipemia may be important. Similarly, a metricthat is indicative of lipemia, but stable with regard to variations inhemolysis also may provide important indications of sample state.

An experiment was designed for artificially creating hemolysis andlipemia samples. Ultraviolet-visible (UV-Vis) absorbance measurementswere carried out using a NanoDrop® 2000c Spectrophotometer (ThermoScientific, Barrington, Ill., USA) and were performed by applying 2 μLof sample on the micro-volume pedestal.

Linear regression models, R2 and coefficients of variation (CV) werecomputed using excel. Hemolysis correction factors and resulting HSscores were calculated using the NanoDrop results.

Sample collection was performed via the following protocol: (1) 1volunteer-plasma sample in EDTA or heparin tube (2) Plasma and red bloodcell (RBC) separation was performed straight away at 2000×g, 15 min, 4degrees Celsius; (3) plasma should be stored at minus 80 degreesCelsius; (4) RBC should be vigorously mixed using a vortex and stored at4 degrees Celsius until further use (5) for 3 volunteers, 20 mL of serumsample (3 vials) was collected under normal protocol (6.5 mL of serumper person minimum), (6) Samples were kept on ice during the wholehemolysis experiment process.

Following sample collection, a hemolysis assay was prepared and measuredas follows:

(1) For each of the 3 volunteers, 500 μL of serum sample with 0.5% RBCcontent was prepared by mixing 497.5 μl serum with 2.5 μL of RBC. Inparticular, RBC1=0.5% hemolysis=497.5 μL PS+2.5 μL RBC.

(2) A serial dilution was prepared as follows (HS=hemolysis sample,PS=pure serum):

RBC2=0.25% hemolysis=100 μL of 0.5% HS+100 μL of PS

RBC3=0.125% hemolysis=100 μL of 0.25% HS+100 μL of PS

RBC4=0.0625% hemolysis=100 μL of 0.125% HS+100 μL of PS

RBC5=0.03125% hemolysis=100 μL of 0.0625% HS+100 μL of PS

RBC6=0.015625% hemolysis=100 μL of 0.03125% HS+100 μL of PS

RBC7=0.007813% hemolysis=100 μL of 0.015625% HS+100 μL of PS

RBC8=0.003906% hemolysis=100 μL of 0.007813% HS+100 μL of PS

RBC9=0.001953% hemolysis=100 μL of 0.003906% HS+100 μL of PS

The dilution series is shown in Table 3.

TABLE 3 Dilution series for a Hemolysis assay Dilution Series 0.25 0.1250.0625 0.03125 0.015625 0.007813 0.003906 0.001953

(3) Measurements with the Nanodrop at wavelengths of A414-385/A660-700were recorded for each sample in the dilution series. The samples werethen stored at −80° C. A measurement of a pure sample (RBC 0) was alsotaken.

A lipemia dilution series was prepared and measured similarly, accordingto the following steps:

(1) For each of the 3 volunteers, 2200 μL of serum sample with 0.8%lipid content was prepared according to L1=2182.4 μL PS+17.6 μLLipofundin (lipofundin MCT 5 g+5 g/100 mL, B. Braun Melsungen Ag,Melsungen, Germany)=0.8% Lipemic sample (0.8% LP)(2) A serial dilution from the 0.8% LP (Original LP) was then preparedas follows

L2=0.4% lipemic sample(LP)=1015 μL of 0.8% LP+1015 μL of PS

L3=0.2% lipemic sample(LP)=875 μL of 0.4% LP+875 μL of PS

L4=0.1% lipemic sample(LP)=600 μL of 0.2% LP+600 μL of PS

(3) Measurements with the Nanodrop at wavelengths of A414-385/A660-700were recorded for each sample in the lipemia dilution series. Thesamples were then stored at −80° C.

Finally, a Hemolysis and Lipemia assay (RBC+LP) was prepared accordingto the following steps:

(1) For each of the 3 volunteers hemolysis+lipemic samples usingdifferent lipemia dilutions of each volunteer and making stepwisedilution for hemolysis as shown below in order to produce samples withvarying levels of % hemolysis (according to the values shown in Table 4)in the presence of different levels of lipemia (0.8%, 0.4%, 0.2% and0.1%):

TABLE 4 Dilution series for hemolysis for samples containing bothhemolysis and lipemia. Dilution Series 0.25 0.125 0.0625 0.031250.015625 0.007813 0.003906 0.001953In detail:

Dilutions A:

RBC+L1A=199 μL of 0.8% LP+1RBC=0.8% lipemia+0.5% HS

RBC+L2A=100 μL of RBC+L1A+100 μL of 0.8% LP=0.25% HS

RBC+L3A=100 μL of RBC+L2A+100 μL of 0.8% LP=0.125% HS

RBC+L4A=100 μL of RBC+L3A+100 μL of 0.8% LP=0.0625% HS

RBC+L5A=100 μL of RBC+L4A+100 μL of 0.8% LP=0.03125% HS

RBC+L6A=100 μL of RBC+L5A+100 μL of 0.8% LP=0.015625% HS

RBC+L7A=100 μL of RBC+L6A+100 μL of 0.8% LP=0.007813% HS

RBC+L8A=100 μL of RBC+L7A+100 μL of 0.8% LP=0.003906% HS

RBC+L9A=100 μL of RBC+L8A+100 μL of 0.8% LP=0.001953% HS

Dilutions B:

RBC+L1B=199 μL of 0.4% LP+1 μL RBC=0.4% lipemia+0.5% hemolysis.

Stepwise dilutions through 0.001953% were prepared as described abovewith regard to Dilutions A.

Dilutions C:

RBC+L1C=199 μL of 0.2% LP+1 μL RBC=0.2% lipemia+0.5% hemolysis.

Stepwise dilutions through 0.001953% were prepared as described abovewith regard to Dilutions A.

Dilutions D:

RBC+L1D=199 μL of 0.1% LP+1 μL RBC=0.1% lipemia+0.5% hemolysis.

Stepwise dilutions through 0.001953% were prepared as described abovewith regard to Dilutions A.

During the experiment it was found that not enough volume was left tohave the dilutions of the last part of the experiment (RBC+L) and stillkeep enough volume of L dilutions to carry out MS analysis (at least 100μL), so the last dilution amount of RBC+L was reduced following way:

RBCL9B,45 μL+45 μL=90 μL  Patient 1,

RBCL9C,50 μL+50 μL=100 μL  Patient 2,

RBCL9D,50 μL+50 μL=100 μL  Patient 2,

RBCL8A,70 μL+70 μL=140 μL  Patient 3,

RBCL9A,35 μL+35 μL=70 μL  Patient 3,

RBCL9B,50 μL+50 μL=100 μL  Patient 3,

RBCL9C,50 μL+50 μL=100 μL  Patient 3,

RBCL9D,50 μL+50 μL=100 μL  Patient 3,

(2) As with the pure hemolysis and lipemia samples, measurements withthe Nanodrop at wavelengths of A414-385/A660-700 were recorded for eachsample in the hemolysis+lipemia dilution series. The samples were thenstored at −80° C.

Measurements performed at the different wavelengths for the differentdilutions are shown in Table 5, Table 6, and Table 7.

Graphics(L1 to L4): y=Δ414-385; x=A385; a=factor

HS=Δ414-385+(factor*A385)

Table 5 shows Nanodrop measurements for samples with hemolysis, but nolipemia.

TABLE 5 Nanodrop measurements for directly evaluating hemolysis withA414-385 and by applying HS correction score. % hemolysis % lipemia A385A414 A414-385 A660-700 HS Score 0 0 RBC 10 0.066 0.120 0.054 0.0040.068328 0.001953 0 RBC 9 0.070 0.124 0.054 0.003 0.06912 0.003906 0 RBC8 0.071 0.130 0.059 0.001 0.074336 0.007813 0 RBC 7 0.070 0.136 0.0660.004 0.08112 0.015625 0 RBC 6 0.074 0.154 0.080 0.002 0.095984 0.031250 RBC 5 0.092 0.194 0.102 0.000 0.121872 0.0625 0 RBC 4 0.098 0.2450.147 0.003 0.168168 0.125 0 RBC 3 0.141 0.400 0.259 0.002 0.289456 0.250 RBC 2 0.212 0.666 0.454 0.004 0.499792 0.5 0 RBC 1 0.388 1.277 0.8890.005 0.972808

FIG. 5 shows a linear increase obtained with increase hemolysis forA414-385 values represented in Table 5, indicating good accuracy forusing A A414-385 as a measurement indicative of hemolysis for samplesdisplaying only hemolysis.

A414-385 values were then measured for samples displaying both hemolysisand lipemia. All samples displayed a linear relationship between theextent of lipemia (in terms of A385 values) and Δ A414-A385 estimation(mean R2=0.996, range R2=0.986-0.999) following the trend line equationΔ A414−A385=a*A385+b (FIG. 6). The angular coefficient, a, wascalculated for all samples and a mean absolute value of |ā|=0.216(CV=2.9%) was obtained.

Samples with the same % hemolysis, but different % lipemia were found tohave different A414-385 (Table 6) values. The HS score, however, wassubstantially the same across the samples. This indicates that foraccurate evaluation on % hemolysis, HS Score should be used instead ofsimple A414-385 measurement.

TABLE 6 UV-Vis absorbance for samples with different lipemia levels andhemolysis levels. The lipemia levels influence the measurement ofA414-385, while HS score appears robust to variations in lipemia %hemolysis % lipemia A385 A414 A414-385 A660-700 HS Score 0.001953 0.8RBC + L A 9 0.503 0.471 −0.032 0.022 0.076648 0.001953 0.4 RBC + L B 90.286 0.28 −0.006 0.009 0.055776 0.001953 0.2 RBC + L C 9 0.181 0.2130.032 0.004 0.071096 0.001953 0.1 RBC + L D 9 0.126 0.171 0.045 0.0030.072216 0.003906 0.8 RBC + L A 8 0.512 0.483 −0.029 0.018 0.0815920.003906 0.4 RBC + L B 8 0.282 0.295 0.013 0.006 0.073912 0.003906 0.2RBC + L C 8 0.176 0.212 0.036 0.004 0.074016 0.003906 0.1 RBC + L D 80.125 0.173 0.048 0.004 0.075

Measurements for evaluating lipemia in the presence of hemolysis werealso evaluated. Table 7 shows measurements of A385 and A660-770 for aconstant lipemia level and varying hemolysis. The A660-700 absorbancemeasurement does not vary substantially with % hemolysis. In contrast,the A385 measurement changes depending on the hemolysis level of thesample despite the % lipemia remaining constant. Accordingly, A660-700may provide a more reliable measurement of lipemia in a sample, robustto variations in hemolysis, than the commonly used A385 measurement.

TABLE 7 Comparison of measurements of A385 and A660-700 for samples withvarying % hemolysis and constant % lipemia % hemolysis % lipemia A385A660-700 0.001953 0.8 RBC + L A 9 0.503 0.022 0.003906 0.8 RBC + L A 80.512 0.018 0.007813 0.8 RBC + L A 7 0.51 0.022 0.015625 0.8 RBC + L A 60.519 0.021 0.03125 0.8 RBC + L A 5 0.534 0.020 0.0625 0.8 RBC + L A 40.554 0.025 0.125 0.8 RBC + L A 3 0.603 0.020 0.25 0.8 RBC + L A 2 0.0920.020 0.5 0.8 RBC + L A 1 0.846 0.024

In certain embodiments, the methods described herein for determining thehemolysis and lipemia levels in a sample may be used as qualitycontrols. For example, samples exhibiting higher levels of hemolysisand/or levels of lipemia may be excluded from analysis. In certainembodiments, measurements of lipemia and hemolysis may be applied ascorrection factors or additional parameters in the predictive model.

Time at Room Temperature Quality Control Marker

In certain embodiments, adequate serum extraction protocols are a keyfor successful measurement and estimation of the disease state of thepatient. Accordingly, the influence of the time that blood samples werekept at room temperature prior to serum extraction on the markers wasevaluated. Blood samples were extracted from 6 volunteers. For eachvolunteer 5 tubes of venous blood were collected and differentextraction time-points were applied to each of the tubes ranging from 30min, 2 h, 4 h, 8 h and 24 h from blood extraction. All samples werefrozen after serum extraction and kept at −80 degrees Celsius for acouple of days. A metabolite extraction method was applied to all thesamples according to a standard protocol. All samples were analyzed as 1analysis set according to the analysis protocols based on, e.g. usingFIA for fatty acid analysis, C18 columns for metabolite analysis, andAmide columns for polar metabolite analysis.

Certain markers measured via the Amide and C18 columns were affected byserum time at room temperature from 2 hours forward. The majority ofaffected markers had a considerable increase after 4 hours at roomtemperature which correlates with our strict sample collection protocolfor prospective sample collection.

The marker S150 was found to be strongly influenced by time at roomtemperature. The marker measurement showed an increase of 23% on averagefor extraction times of 2 h in comparison with extraction times of 30min. An exponential increase was observed as a function of time toextraction for times up to 24 h.

In one embodiment, based on measurements of S150 and mass spectrometricmeasurements on over 1000 individual stored samples an estimated valueof 15 μg/mL was determined as a cut-off for exclusion of the sample fromfurther analysis. S150 values higher than 15 μg/mL correlated withconcentration measurements for other markers that were either increasedor decreased from their normal values and would therefore lead toinaccurate sample classification.

Sample Collection Quality Control Marker

In one embodiment, a comparison of different sample sources andcollection methods indicated that S10 is a marker indicating a certaintype of collection protocol used. In certain embodiments, under normalcollection protocols the values of S10 are 10 times lower than underinappropriate sample collection protocol using collection tubes. Asappropriate collection is prerequisite for accurate metabolicmeasurements, this marker may be used as an indicator for samplecollection that can result in inaccurate classification. Accordingly, incertain embodiments, measurement of the S10 marker may be used as aquality control measure.

Markers for Icterus Measurements

Icterus may cause an excess of bilirubin pigment or bilirubin complexesin the bloodstream. The bilirubin pigment or bilirubin complexes mayinterfere with spectrophotometric measurements.

Three molecules from bile acid pathway (S52, S61 and S62) can bemeasured with the systems and methods described herein. These moleculesare believed to connected to jaundice and liver dysfunction, which canresult in bilirubin accumulation.

Internal Standards for Mass Spectrometry Measurements

In certain embodiments, methods and protocols that improve the accuracyand reproducibility of the mass spectrometry measurement itself are alsoimportant to employ.

For example, before measuring an experimental sample, it is necessary tostabilize the mass spectrometry equipment by running 5-10 qualitycontrol samples. Mass spectrometry equipment is prone to giving falseresults before stabilization and, accordingly, the first 5-10 qualitycontrol sample measurements should be discarded. Additional qualitycontrol and blank samples need to be run during after every 10 analysissamples. Moreover, additional quality control can be provided by runningall analysis samples in duplicate. For example, samples that have a CV%>20 between duplicate samples for more than 20% of the markers areexcluded from the further analysis. The sample preparation procedure isrepeated for these samples.

In certain embodiments, isotopically labelled internal standards areuseful to include in order to enable accurate and reproduciblequantification of a molecule of interest (e.g. a marker). For example,S192 (3Me-Glutaryl Carnitine) is an important marker in panels, but waschallenging to measure without a proper internal standard.

An isotopically labelled internal standard may be a synthetic equivalentof the molecule of interest that is modified by replacing specific atomsby their isotopes. A known concentration of the standard is thenartificially added to the sample to be analyzed (e.g. a serum sample)and extracted and analyzed alongside the biological molecule ofinterest. As the concentration of the synthetic molecule is known thenthe concentration of the biological molecule can be calculated with thehelp of fitting the synthetic one to a calibration curve of the method.

There were 11 metabolites (listed in Table 8) that do not havecommercially available internal standards. Custom standards weresynthesized for these molecules.

TABLE 8 List of molecules that required custom synthesis a correspondinginternal standard. IS Marker AMIDE S325 CUSTOM SYNTHESIS S179 CUSTOMSYNTHESIS Marker C18 S103 CUSTOM SYNTHESIS S168 CUSTOM SYNTHESIS S295CUSTOM SYNTHESIS S166 CUSTOM SYNTHESIS S175 CUSTOM SYNTHESIS S227 CUSTOMSYNTHESIS S285 CUSTOM SYNTHESIS S3 CUSTOM SYNTHESIS S192 CUSTOMSYNTHESIS

Biomarker Discovery Approach

An example biomarker discovery approach, which was used to discover thelist of markers provided herein is also included in the following.

In the embodiment, five different approaches (untargeted profiling ofmetabolites, targeted detection of lipids (e.g., fatty acids),utilization of Biocrates targeted analysis kit, utilization of targetedmetabolite panel offered by Metabolon Inc. and literature and databasesearch over metabolites that have been connected to cancer and itsprogression) and 4 different analytical platforms (LC-MS on Agilent QTOFwith C18 50 mm column and Amide column, LC-MS on AB Sciex QTRAP with C18100 mm column and FIA (flow injected analysis) injection on ABSciexQTRAP) were used to identify significant markers.

A first step in the example assay development process as describedherein was biomarker discovery using 5 different approaches (e.g.,untargeted profiling with LC-MS on Agilent QTOF, e.g., utilization ofBiocrates targeted analysis kit on ABSciex 5500 QTRAP, e.g., outsourcingof lipid analysis from Lipotype GmbH, e.g. FIA injection on ABSciex TQ4500MD, e.g. targeted profiling by Metabolon Inc.) to pool outsignificant markers. A total of 505 samples were analyzed of which 450belonged to CRC analysis panel and 55 were lung cancer samples wereanalyzed across those 5 approaches.

A serum-based global metabolic profiling test was performed to detectbiomarkers that are indicative of certain health state. The initialbiomarker discovery study was based on using untargeted profilingtechnique utilizing liquid chromatography coupled mass spectrometryequipment for screening total of 415 samples. This type of screeningoption allows detection of and provides intensity values for hundreds ofdifferent small molecules present in human sera. Together withappropriate statistical tools, a set of significant markers wereidentified.

The biomarker discovery process also used additionally a targetedapproach by analyzing 202 samples. In certain embodiments, AbsoluteIDQp180 Kit was used, produced and provided by Biocrates Life Sciences AG.AbsoluteIDQ p180 Kit can be used for targeted detection andquantification of 186 pre-defined molecules belonging to differentmetabolite and lipid classes. A sample set used for this part of thebiomarker discovery partially overlapped with global profilingexperiments that were performed previously and partially employed newsamples.

Lipid analysis was outsourced from Lipotype GmbH, Dresden, Germany. Thesamples were provided to Lipotype GmbH, who performed the samplepreparation and analysis. Lipotype GmbH provided a list of lipids withsemi-quantitative values as result. A sample set of 120 samples was usedin this targeted profiling method. This sample set overlapped with thesamples used in the untargeted profiling approach.

A search into published literature and biological pathways was alsoperformed to find markers that might be influenced by cancerdevelopment. The most significant finding from this search was 6polyunsaturated fatty acids—PUFA molecules. Structurally, the moleculesresemble very long chain (28 carbon) mimetics of the resolvins andprotectins, containing multiple double bonds and at least two hydroxylgroups. FIG. 1 shows a structure of the 6 molecules.

In addition to all the previous discovery phase another outsourcingoption was used by performing targeted profiling experiment on 120samples and 800 identified putative identification markers designed into1 panel by Metabolon Inc. This sample set overlapped with the samplesused in untargeted profiling approach.

Significant markers were identified from the 5 different discoveryapproaches and combined using feature selection and statistical patternrecognition methods as described herein.

A second step in the example process was optimization of analyticalplatforms for the significant markers identified. A first analyticalpanel of 18 markers generated was tested with new set of 369 samples forverifying the performance. After combining significant markers from allthese 5 discovery options another set of optimization and significantfeature selection experiments were performed to verify and fix the panelto the list shown in FIG. 1. After additional identification, methodoptimization and a second phase of feature selection a panel of 78markers for LC-MS based analysis that has been divided into 3 methodsaccording to separation column (as specified above) was obtained. Anadditional 6 markers to be measured with FIA-MS analysis was also found.Overall, 84 markers to be measured using distinct analysis methods werefound (FIG. 1 and Table 1). Results based on 678 new samples and 30metabolic markers are presented in further paragraphs on this document.

Additional Panel Study Using 30 Metabolic Markers

In an example study, the performance of a 30 metabolic marker panelsbased on a subset of the markers listed in Table 1 and Table 2 wasfurther evaluated using 682 retrospective samples from a population ofpatients from Spain, Ukraine, Russia, UK and USA. Samples were obtainedfrom following sources (Hospital Victoria Eugenia, Sevilla, Spain,IDIBAPS biobank Barcelona Spain, Asterandbio biobank USA (withcollection from Russia and Ukraine, Biosevere USA, Biooption USA, FolioUSA, Promeddex USA and Tissuesolution Glasgow UK). Patients with allstages of colorectal cancer, individuals without diseases of the colonas verified by colonoscopy, additional disease controls, and a number ofpatients with adenomatous polyps were included.

General sample collection rules were as follows. Blood from adenoma, asubset of CRC patients, and control subjects had been drawn priorcolonoscopy. Blood was drawn for a subset of CRC patients prior tostarting any cancer specific treatment. Cancer diagnosis was confirmedhistologically from the surgical specimen. A subset of the controls usedwas not verified to be adenoma free by colonoscopy, but they werebelieved to be cancer free. All subjects participating had neither apersonal history of HIV, HBV or HCV, nor previous history of cancer.Serum was extracted and frozen down within 4 hours from drawing bloodfrom the patients. Samples were collected under fasting conditions andstored at −80° C.

Serum samples were stored at −80° C. until thawed for analysis. Sampleswere only thawed once. Samples were kept on ice until extraction processthat was performed at room temperature. Serum samples were prepared forMS analysis by first sequentially extracting serum with a 3:1 volume ofice cold methanol. Samples were incubated and centrifuged at 4° C. for10 min at 3500 rpm and the organic layer was removed and transferred toa new tube (extract A). Extract A was then completely evaporated undernitrogen and reconstituted in original sample volume of ACN:H2O 5:95(extract B). All extracts were either stored at −80° C. or analyzedstraight away with MS.

A panel of 30 different markers was measured with 3 differentmethods-FIA based direct infusion injection for analyzing fatty acidmarkers, LC-MS C18 50 mm column based method for metabolite detectionand LC-MS Amide column based metabolite detection method. In particular,30 MRM transitions were analyzed with the 3 different MS methods.Fifteen markers were measured using conventional reverse phasechromatography, 9 polar compounds were measured in an amide columnmethod and 6 markers were measured via direct injection method with FIA.A list of the metabolites along with the corresponding measurementmethod is shown in Table 9. These markers are a subset of the markerslisted in the table shown in Table 1. These metabolites belong to 6different major chemical classes (such as amino acids and theirderivate, vitamin derivate, carboxylic acids, dipeptides,micronutrients, nucleosides, carnitines, lipids and fatty acids), whichare located in important metabolic pathways (e.g., TCA cycle, amino acidmetabolism, glycolysis, lipid metabolism, Krebs cycle), in both positiveand negative ionization modes.

TABLE 9 List of markers S1 C18 S63 C18 S100 C18 S103 C18 S109 C18 S147C18 S193 C18 S227 C18 S69 C18 S166 C18 S3 C18 S192 C18 S76 C18 S175 C18S285 C18 S10 C18 S150 C18 S168 C18 S295 C18 S125 Amide S132 Amide S153Amide S236 Amide S261 Amide S49 Amide S110 Amide S78 Amide S111 AmideS179 Amide S325 Amide PUFA 446 FIA PUFA 448 FIA PUFA 450 FIA PUFA 464FIA PUFA 466 FIA PUFA 468 FIA

Two calibration curves were prepared to quantify all markers of thepanel. Curve 1 contains 6 calibration levels and curve 2 contains 8calibration levels. Calibrators were prepared freshly every day.

SeraSub® was used as a blank matrix. SeraSub is a synthetic polymer inbuffered solution that is physically equivalent to serum and plasma withrespect to specific gravity, viscosity and osmolality.

Standard 6 (STD 6) and standard 8 (STD 8) from curves 1 and 2respectively, were used to build the other standards by serialdilutions.

In the present example study, all samples, quality control samples andblank standards were prepared as one analysis set and analyzed in oneanalysis run. Blank samples and QC samples were analyzed every 10samples for evaluating stability of the system over a long run andapplying normalization for the samples. All samples were analyzed induplicates. A CV<20% QCs run between all analysis sets performed indifferent days was considered acceptable. Raw data was transformed intoarea values using the MultiQuant software tool from ABSciex. MultiQuantsoftware is used for evaluating the integrity of the peaks generated viaanalysis, and for integrating peak values in order to obtain absoluteconcentration. Stable isotope-labeled internal standards were used for15 markers measured via reverse phase chromatography, and 9 markersmeasured via amide column for absolute quantification purposes, and formonitoring instrument performance.

In the embodiment, data analysis was performed using a random forestclassifier, based on the remaining 30 markers. In addition to themeasured concentration of the markers, additional features weregenerated by taking the ratios of the measurements between individualmarkers. The resulting 400+ratios were evaluated for their performanceand correlation, and the best combinations were used for training themodel. A subset of 317 samples was used to generate the training setalgorithm, and 365 samples were used in the testing study. A balancedtraining dataset independent from physical factors such as age, race orgender was built. The three conditions that were met in the trainingdataset have balanced distributions regarding age, gender and race. Thedemographic and clinical parameters of the subjects included in thetraining study are outlined in Table 10.

TABLE 10 Clinical data of the sample set used for training the algorithmCRC Polyp Stage Stage Stage Non Stage I II III IV Unknown AA AA UnknownAge   <60 16 14 18 8 1 33 21 8 60-70 10 16 20 9 3 36 19 8 70-80 5 4 6 65 8 15 1 >=80 0 1 4 0 1 0 1 1 Gender Female 14 20 17 14 6 36 28 9 Male17 15 31 9 4 41 28 9 Race Black 0 0 0 0 0 0 1 0 Caucasian 31 34 48 23 1076 55 18 Hispanic 0 1 0 0 0 0 0 0 Other 0 0 0 0 0 1 0 0

Monte-Carlo cross-validation was performed over the full sample setaccording following parameters:

Random Forest Classifier

-   -   Ntree=1000    -   Mtry=sqrt(#Markers)    -   Monte Carlo Cross Validation (20 fold)

To confirm the performance of training algorithm an independent patientset was used. The testing set was comparable to training set with regardto age, gender and stage distribution for CRC and polyps. Thedistribution of the testing set is shown in Table 11.

TABLE 11 Clinical data of the sample set used for testing the algorithmCRC Stage Stage Stage Stage Un- Con- I II III IV known Polyp trol Age  <60 14 10 11 7 2 55 55 60-70 7 12 15 6 2 49 51 70-80 4 6 3 5 3 1817 >=80 1 0 3 0 2 2 4 Gender Female 11 15 12 13 3 58 59 Male 15 13 20 56 66 69 Race Other 0 1 0 0 0 4 0 Caucasian 26 27 32 18 9 120 128 SUM 113124 128

Using the model threshold determined in the training study, the panelperformance was confirmed in the testing set of 365 CRC, polyp andcontrol patients. An AUC of 92% and a CI in the range (88.45%, 95.8%)were determined. Sensitivity and specificities of 87% and 83% were alsodetermined, as shown in FIG. 7. There was no significant difference indetection rate by age or gender. Sensitivity for detecting onlycolorectal cancer regardless of the stage was 87 and sensitivity ofdetecting cancer of different stages ranged from 82-85% in early stageto 89-94% in later stages, as shown in FIG. 8. Adenomatous polyps weredetected with general sensitivity of 83% (FIG. 7).

Certain features—corresponding to either ratios of two markers orparticular markers measured in isolation—were found to be significant indistinguishing between patients with CRC and the control group, orbetween PP and the control group, when used in isolation.

The features relevant for CRC detection are listed in Table 12. Thefeatures relevant for detecting adenomatous polyps are listed in Table13. The features based on a ratio of two markers as opposed to ameasurement of a single marker appear to perform particularly well. Itis important to note that these values represent univariate performance,meaning they are values representing the single feature performance(single marker in this sense means also a ratio), not the final panel.Markers in the panel that are not highly indicative as individualmarkers still contribute to the performance of the classification modelthat uses the overall panel. Certain molecules referred to in Table 12and Table 13 are represented in FIG. 1 and FIG. 2.

FIG. 9 shows box plots of markers or ratios of markers in Table 12 thathave high significance for detecting colorectal cancer. Experimentalvalues for each marker or ratio of markers are shown for a populationwith colorectal cancer and a control population. The selectivity isqualitatively observed as the difference in distributions between thecontrol population and the population having colorectal cancer.Calculated measures of selectivity are listed in Table 12. FIG. 10 showsbox plots of markers or ratios of markers in Table 13 that have highsignificance for detecting adenomatous polyps as compared to a control.Experimental values for each marker or ratio of markers are shown for apopulation with adenomatous polyps and a control population. Theselectivity is qualitatively observed as the difference in distributionsbetween the control population and the population having adenomatouspolyps. Calculated measures of selectivity are listed in Table 13.

TABLE 12 Significant markers for distinguishing between CRC vs Controlranked based upon their univariate performance Name Kappa SensitivitySpecificity AUC Accuracy PUFA 446/S192 0.55 0.72 0.83 0.83 0.77 PUFA468/S192 0.54 0.74 0.80 0.82 0.77 S49/S236 0.52 0.76 0.76 0.83 0.76 PUFA446/S103 0.51 0.73 0.77 0.81 0.75 PUFA 468/S285 0.50 0.77 0.73 0.81 0.75PUFA 446 0.50 0.68 0.82 0.79 0.75 PUFA 468/S76 0.49 0.67 0.83 0.79 0.75PUFA 468 0.47 0.68 0.78 0.77 0.73 S3/S236 0.46 0.70 0.76 0.80 0.73 PUFA468/S103 0.46 0.69 0.76 0.80 0.73 PUFA 448 0.46 0.64 0.81 0.78 0.73S175/S236 0.45 0.70 0.75 0.80 0.72 S1/S236 0.45 0.68 0.76 0.78 0.72 PUFA450/S103 0.45 0.67 0.78 0.80 0.72 S132/S236 0.44 0.68 0.77 0.77 0.72

TABLE 13 Significant markers for distinguishing between PP vs Controlranked based upon their univariate performance Name Kappa SensitivitySpecificity AUC Accuracy S153 0.56 0.77 0.78 0.80 0.78 S69/S153 0.550.80 0.75 0.82 0.78 S109/S153 0.55 0.72 0.83 0.83 0.78 S227/S63 0.450.76 0.69 0.78 0.72 S109/S63 0.44 0.64 0.81 0.75 0.72

1. A method comprising: (a) measuring, by mass spectrometry, a level ofeach of a plurality of species in a biological sample obtained from abody fluid of a human subject, wherein each of the plurality of speciesis at least one of a metabolite and a fatty acid and the plurality ofspecies comprises:

and 3Me-glutaryl carnitine

(b) determining a ratio of the measured level of PUFA 446 and themeasured level of S192; and (c) determining at least one of a presenceof, a stage of, and a risk of colorectal cancer in the human subjectbased, at least in part, on the ratio of the measured level of PUFA 446and the measured level of S192. 2-4. (canceled)
 5. The method of claim1, wherein the plurality of species comprises one or more members inaddition to PUFA 446 and S192 selected from the group consisting of thespecies listed in Table 12 and Table
 13. 6. The method of claim 1,wherein the plurality of species comprises all of the species listed inTable 12 and 13 and step (c) comprises determining at least one of apresence of, a stage of, and a risk of colorectal cancer in the humansubject based, at least in part, on measured values for the ratios ofspecies listed in Table 12; and the method comprises (d) determining atleast one of a presence of, a stage of, and a risk of adenomatous polypsin the human subject based, at least in part, on measured values for theratios of species listed in Table
 13. 7. The method of claim 1, whereinthe measuring step comprises measuring the level of each of theplurality of species using a LC-MS, GC-MS, DESI, or DART technique. 8.The method of claim 1, wherein step (c) comprises: determining at leastone of a presence of, a risk of, and a stage of colorectal cancer based,at least in part, on a ratio of the measured level of a polyunsaturatedfatty acid of the plurality of species and the measured level of anotherof the plurality of species being lower than a representative ratio fora control population.
 9. The method of claim 8, wherein thepolyunsaturated fatty acid is a species listed in FIG.
 1. 10. The methodof claim 1, comprising: determining at least one of a presence of, arisk of, and a stage of colorectal cancer further based, at least inpart, on a ratio of the measured level of choline (S49) and the measuredlevel of N1,N12-diacetylspermine (S236) being lower than arepresentative ratio for a control population.
 11. The method of claim1, wherein at least one of the plurality of species is selected from thegroup consisting of:

12-15. (canceled)
 16. The method of claim 1, wherein the biologicalsample comprises serum.
 17. The method of claim 1, wherein thebiological sample is serum, plasma, urine, saliva, whole blood, a driedblood spot, or a dried serum spot. 18-19. (canceled)
 20. The method ofclaim 1, comprising: introducing at least a portion of the biologicalsample into a C18 50 mm column, a C18 100 mm column, or an amide columnto determine a quantification of metabolites, lipids or polar metaboliccompounds, respectively, of the plurality of species.
 21. The method ofclaim 1, comprising: introducing at least a portion of the biologicalsample into a mass spectrometer by FIA based direct infusion injectionto measure the level of a polyunsaturated fatty acid of the plurality ofspecies.
 22. The method of claim 1, comprising measuring a stableisotopically labeled reference standard. 23-25. (canceled)